1. Field of the Invention
This invention relates to a method of obtaining antibodies and, more specifically, this invention relates to a method of obtaining an antibody which is highly specific to glycosylated hemoglobin and substantially free of cross-reactivity with non-glycosylated forms of hemoglobin.
2. Description of the Prior Art
Many antigenic molecules which are clinically important are not subject to determination by immunoassay due to their molecular structure. Specifically, some antigens comprise large molecules having many antigenic sites and, as a result, antibodies produced from such antigens may exhibit a high degree of cross-reactivity with respect to various species of antigens, and thus are of only limited utility in immunoassay techniques.
Hemoglobin Alc (HbAlc) has an amino acid structure which is identical to that of the HbAo form of hemoglobin, except for the presence of a glucose moiety attached to the NH.sub.2 -terminus valine in the .beta.-chain. The transformation of HbAo to HbAlc is a continuous process which is a function of blood glucose concentration. The level of HbAlc in a blood sample is an indication of an individual's carbohydrate metabolism.
Normal adults have about 90% of their total hemoglobin as HbAo, 2-3% as HbAla and HbAlb, and 3-6% of their total hemoglobin as HbAlc. However, the level of HbAlc in juvenile and maturity onset diabetics ranges from about 6% to about 15%. The level of HbAlc in hypoglycemic patients is correspondingly less than about 3%.
The quantification of the HbAlc level in diabetic patients is a useful means of assessing carbohydrate tolerance as well as adequacy of control. One prior method of determining the HbAlc level in a serum sample is the well-known column chromotography method wherein the sample is eluted through a column. Glycosylated hemoglobin is eluted prior to nonglycosylated hemoglobin.
However, the results of prior art column chromotography methods are sensitive to many variables, specifically including temperature, column length and ionic strength effects encountered in the column. Prior column methods require pre-separation steps, are not homogeneous, and are relatively nonspecific because they measure HbAo, HbAla, HbAlb, and HbF in addition to HbAlc.
Prior methods of producing antibodies to glycosylated hemoglobin for use in immunoassay procedures involve separation of glycosylated hemoglobin from serum, and production of antibodies by injection of the glycosylated hemoglobin fraction into a host animal. Resulting antibodies are then harvested and labeled, as with a radioactive label, and utilized in suitable assay methodologies, such as RIA.
Exemplary of such prior methods is the work of Cerami and co-workers described in U.S. Pat. No. 4,247,533 (Jan. 27, 1981) and in Javid et al, "Immunologic Characterization and Qualification of Haemoglobin A.sub.lc ", British Journal of Haemotology, Vol. 38 at 329-337 (1978).
Such prior assay methods have generally been unsuccessful, since the hemoglobin molecule is very large, having many antigenic sites, with only minor structural differences between glycosylated and nonglycosylated hemoglobin. Hence, prior assay methodologies have generally been characterized by low specificity due to cross-reactivity of antibodies produced by injection of HbAlc with various forms of nonglycosylated hemoglobin. See Chou et al, "Development of a Laser Nephelometric Method for the Quantitation of Human Glycohemoglobins", Analytical Letters, Vol. 14 (B13) at 1071-1087 (1981).